Magnesium (Mg) chelatase is a heterotrimeric enzyme complex that catalyzes a key regulatory and enzymatic reaction in chlorophyll biosynthesis, the insertion of Mg(2+) into protoporphyrin IX. Studies of the enzyme complex reconstituted in vitro have shown that all three of its subunits, CHL I, CHL D, and CHL H, are required for enzymatic activity. However, a new T-DNA knockout mutant of the chlorina locus, ch42-3 (Chl I), in Arabidopsis is still able to accumulate some chlorophyll despite the absence of Chl I mRNA and protein. In barley (Hordeum vulgare), CHL I is encoded by a single gene. We have identified an open reading frame that apparently encodes a second Chl I gene, Chl I2. Chl I1 and Chl I2 mRNA accumulate to similar levels in wild type, yet CHL I2 protein is not detectable in wild type or ch42-3, although the protein is translated and stromally processed as shown by in vivo pulse labeling and in vitro chloroplast imports. It is surprising that CHL D accumulates to wild-type levels in ch42-3, which is in contrast to reports that CHL D is unstable in CHL I-deficient backgrounds of barley. Our results show that limited Mg chelatase activity and CHL D accumulation can occur without detectable CHL I, despite its obligate requirement in vitro and its proposed chaperone-like stabilization and activation of CHL D. Thus, the unusual post-translational regulation of the CHL I2 protein provides an opportunity to study the different steps involved in stabilization and activation of the heterotrimeric Mg chelatase in vivo.